Guide to the Disruption of Biological Samples - 2012

When developing a disruption scheme, it is prudent to define the
characteristics of the desired homogenate and then select the methods,
reagents, and tools that will help to meet the objectives. There
is a vast selection of chemistries and tools that have been endlessly
combined to disrupt samples. Detergents, chaotropes, and lytic
enzymes are often effective for lysing cells and tissues, many times
alone, but also when combined with mechanical methods.
Mechanically disrupting samples using homogenizers, which can be grouped
into those that grind, shear, beat, and shock, is commonplace when
chemical methods alone are insufficient. The examination of
methods used to homogenize samples has shown that effectiveness is
directly related to the nature of the sample. Samples that start
with small particles, such as bacterial cultures, are most effectively
disrupted by ultrasonication, but that same method is the poorest for
solid muscle. In such cases, samples must first be disaggregated
into smaller particles prior to processing. Methods which rely on
a single processing step, such as with the high throughput homogenizers,
can yield very good sample disruption, but they do not match two-step
processes that breakdown samples in a series of steps. The need to
process large numbers of samples may require a trade-off with the
effectiveness of homogenization.

Disruption is an early and fundamental step in any research
which involves analyzing, separating, or isolating some
component from an intact sample. This includes the
isolation/harvesting of cellular components or quantification of
RNA, DNA, proteins, and analytes. Both chemical and
mechanical/physical methods are available for disruption, with
chemical methods being preferred for many sample types (e.g.,
E. coli and cultured cells). However, many microorganisms,
intact tissues, solid specimens (e.g., seeds), and heavily
encased samples are not effectively disrupted chemically.
With chemically resistant samples, mechanical and physical
methods that rely on grinding, shearing, beating and shocking
can be used. Mechanical homogenizers, manual homogenizers,
mortar and pestles, sonicators, mixer mills, and vortexers are
several of the more common tools used for mechanical and
physical disruption.

Sample disruption, or homogenization, is often scantly
detailed in protocols even though it does have significant
impact on the end results of a process. Many research
articles will simply state that a sample was “homogenized” in a
defined buffer, without specifying what type of homogenizer was
employed. When specific homogenizers are mentioned, such
as the Dounce or bead beater, little additional information is
provided to detail its use. Consequently, methods used for
sample disruption are also not necessarily well understood.
Indeed like many well established lab processes,
homogenization methods are passed on from researcher to
researcher like inheritable family treasures, with little effort
expended to decipher the process itself. This leads to
significant variation in methodology between laboratories.
Being fair, the impact of homogenization on an experiment may be
minimal, but at times the choice of tools, chemistries, and
their method of use may have a significant impact on the
outcome.

Where possible, chemical disruption may be the preferred
method, such as lysing E. coli with SDS for plasmid
isolation, but it may also introduce unwanted molecules into
the lysate. Though useful for nucleic acid isolation,
detergents and chaotropes may certainly denature proteins
making their application to protein purification
impractical. The same is true for the addition of
lytic enzymes, which in the case of protein purification,
must be subsequently removed. If chemical disruption
is impractical or simply does not work, then mechanical and
physical disruption of samples is the alternative.

Mechanical/physical methods for disrupting samples include
grinding, shearing, beating, and shocking. Grinding, which is
done with such tools as a mortar and pestle, involves applying force
downward on a sample in conjunction with a separate tangential
(i.e., rotating) force. Shearing is like that of a blender
where a force is tangentially applied to a sample. Directly
impacting a sample with a ball or hammer is beating. Shock is
similar to beating, but there is no physical implement contacting
the sample, just shockwaves. At times it is difficult to
discern between the different forces that relate to each method.
For instance, grinding is a combination of shearing and beating, but
for the sake of simplicity we will segregate the different tools
into these categories.

In practice, scientists mix and match disruption methods to meet
their needs. Though an ideal disruption method would require
only a single step, it is quite common to see two or more methods
being used in tandem to obtain the desired result. For
instance, the isolation of subcellular fractions could first involve
cutting a tissue with scissors, followed by course shearing with a
handheld homogenizer, and then a final dissociation with a glass
Dounce homogenizer. If any one of these steps is omitted, then
the degree of homogenization would be reduced.

There are several methods we employ to evaluate homogenization
methods which will be highlighted in this guide. First,
homogenization releases cytosolic enzymes and several of these can
be assayed and used as a relatively measurement of sample
disruption. Lactate dehydrogenase is routinely used in our
laboratories for this assessment. Similarly, measuring soluble
protein can be used as an indicator of homogenization efficiency.
Microscopic observation of samples also provides useful information
on the extent by which samples are homogenized.